CN101712866A - Nanometer europium fluorescent particle with performance of visible light excitation, preparation method and application thereof - Google Patents
Nanometer europium fluorescent particle with performance of visible light excitation, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of strong fluorescence nanometer europium fluorescent particles with performance of visible light excitation and the application thereof in biological detection technology thereof. Firstly, an amino silicone alkylating agent is utilized for forming a fluorescent precursor with a ternary europium complex excited by a visible light, and then the fluorescent precursor and silicon ester are polymerized in a water-in-oil type microemulsion to generate the nanometer europium fluorescent particles with the surface provided with active groups. The nanometer europium fluorescent particles have strong visible excitation peaks, the fluorescent lifetime is about 390 microseconds, and the fluorescence quantum yield can reach 66%. The influence of the short-lived background fluorescence can be effectively eliminated when the nanometer europium fluorescent particles are used as biological fluorescent markers for time-resolved fluorescence bioassay, the light stability of the markers can be remarkably enhanced, and the light injury to biological samples is reduced by adopting visible light excitation, and the invention has important application prospect in the fields of chemical and biological detection of time-resolved fluorescence immunoassay, living cell, biological staining and time-resolved fluorescence microscope image analysis, and the like.
Description
Technical field
The present invention relates to a kind of nanometer europium biological marker material of excited by visible light, specifically a kind of preparation and application in the time resolved fluorescence biological detection thereof that can be used for the nanometer europium fluorescent particle with excited by visible light character of biomolecular labeling.
Background technology
Rare earth (europium and terbium) fluorescence complex has very special photoluminescent property, as long fluorescence lifetime, and the emmission spectrum of big Stokes displacement and point.Based on these features, obtained great progress with the serve as a mark time-resolved fluorescent immunoassay technology of thing of rare-earth fluorescent title complex over nearly 20 years, in the practice of medical science and life science and research, bringing into play more and more important effect.In recent years, time resolved fluorescence bio-imaging analytical technology based on the rare-earth fluorescent title complex also becomes a kind of useful instrument, obtained successful application (document 1:K.Suhling at aspects such as the visual mensuration of biomolecules and environment pathogeny microorganism detection, P.M.W.French, D.Phillips, Photochem.Photobiol.Sci.2005,4,13; Document 2:R.Connally, D.Veal, J.Piper, Microsc.Res.Techniq.2004,64,312).The great advantage of time resolved fluorescence bioassay technique is by introducing suitable time of lag after pulse excitation, eliminate the interference of various short life background fluorescences in the various light or organisms samples at random fully, measuring sensitivity thereby greatly improve.
Because the fluorescence radiation of most europiums, terbium coordination compound will pass through intramolecularly triplet energy state transmittance process, it excites window to be limited near ultraviolet region (document 3:F.J.Steemers less than 370nm, W.Verboom, D.N.Reinhoudt, E.B.V.D.Tol, J.W.Verhoeven, J.Am.Chem.Soc.1995,117,9408), this high-octane short wavelength's exciting light not only has certain damaging action to biological sample, and the light stability of marker itself is also had remarkably influenced.Therefore, it is significant to develop a kind of rare-earth fluorescent probe of excited by visible light.Europium complex (document 4:Y.Bretonniere, M.J.Cann, D.Parker, R.Slater, Chem.Commun.2002,1930 that at present existing several research groups have developed several long-wavelength excitations; Document 5:J.H.Yu, D.Parker, R.Pal, R.A.Poole, M.J.Cann, J.Am.Chem.Soc.2006,128,2294; Document 6:R.Pal, D.Parker, Chem.Commun.2007,474; Document 7:M.H.V.Werts, M.A.Duin, J.W.Hofstraat, J.W.Verhoeven, Chem.Commun.1999,799; Document 8:C.Yang, L.M.Fu, Y.Wang, J.P.Zhang, W.T.Wong, X.C.Ai, Y.F.Qiao, B.S.Zou, L.L.Gui, Angew.Chem.Int.Ed.2004,43,5010; Document 9:S.M.Borisov, O.S.Wolfbeis, Anal.Chem.2006,78,5094), but because these title complexs have existed in water-insoluble, the polar solvent drawbacks limit such as instability or quantum yield are low its application in biomarker.
The rare-earth fluorescent title complex another one shortcoming that thing uses that directly serves as a mark is anti-photobleaching ability, and this also is the common defects of all molecular marked compounds.Along with the development of nano biological analytical technology, various functional nano fluorescent materials occur in succession, and are widely used in the biometric techniques field such as unit molecule in situ detection, fluorescent microscope detection of biomacromolecule.The nano fluorescent bioprobe makes its fluorescent signal obtain amplifying owing to can coat a large amount of fluorescence molecules, significantly improves the sensitivity of mensuration, and the light stability of fluorescence molecule also can significantly strengthen under the protection of skin coating matrix.The nano fluorescent bioprobe of having developed at present mainly contains: (1) semiconductor nano (claiming quantum dot again); (2) (class is a high molecule plastic substrate nano fluorescent grain to the composite Nano fluorescent grain of bag fluorescence molecule in, another kind of is the nanometer silica gel particle of the various fluorescent substances of interior bag) (document 10:W.H.Liu, M.Howarth, A.B.Greytak, Y.Zheng, Nocera, D.G.A.Y.Ting, M.G.Bawendi, J.Am.Chem.Soc.2008,130,1274; Document 11:X.J.Zhao, L.R.Hilliard, S.J.Mechery, Y.P.Wang, R.P.Bagwe, S.G.Jin, W.H.Tan, Proc.Natl.Acad.Sci.USA 2004,101,15027), but these fluorescent particles still exist light-emitting flash, labeling process complexity when being used for biological assay, fluorescence molecule easily leaks and serious problems such as background fluorescence interference.
Recently, several composite Nano fluorescent particles based on europium, terbium coordination compound have been developed and have been used to highly sensitive time resolved fluoro-immunoassay and imaging detection (document 12:Y.Xu, Q.G.Li, Clin.Chem.2007,53,1503; Document 13:P.Huhtinen, M.
O.Kuronen, V.Hagren, H.Takalo, H.Tenhu, T.
H.
Anal.Chem.2005,77,2643; Document 14: Yuan Jingli, Tan Mingqian, Ye Zhiqiang, Wang Guilan, a kind of functional nano rare-earth fluorescent particulate and preparation and application, Chinese invention patent, the patent No.: ZL02144517.6; Document 15: Yuan Jingli, Tan Mingqian, Hai Xiaodan, Wang Guilan, beta-diketon-trivalent europium complex namo fluorescence probe and preparation and application, Chinese invention patent, the patent No.: ZL03133857.7; Document 16:J.Wu, G.L.Wang, D.Y.Jin, J.L.Yuan, Y.F.Guan, J.Piper, Chem.Commun.2008,365), yet these nano rare earth fluorescent particles still can't avoid using high-octane burst of ultraviolel light source, therefore are restricted in some Application Areass.
Summary of the invention
The purpose of this invention is to provide a kind of hyperfluorescence nanometer europium fluorescent particle that has the excited by visible light performance and can be used for biomarker, and it is measured as the time resolved fluorescence micro-imaging that fluorescent marker is used for time-resolved fluorescent immunoassay and complex environment sample pathogeny microorganism.
Technical scheme of the present invention is as follows:
At first with four tooth beta-diketon class ligands and the reaction of aminosilane reagent generation covalent attachment, again with EuCl
3And 2-(4-N, N-diethylbenzene amido)-4,6-two (3,5-dimethyl pyrazole base)-1,3, the fluorescence precursor of excited by visible light is made in 5-triazine (DPBT) reaction, then this precursor and silicon ester is caused copolymerization with the silanization monomer that has active group amino or sulfydryl with ammoniacal liquor, has the nanometer europium fluorescent particle that as seen excites by force with the biomarker function thereby prepare.The structural formula of used four tooth beta-diketon class ligands and DPBT is as follows:
Wherein R is=C
6F
5Or fat hydrocarbon substituting group C
nH
2n+1, C
nF
2n+1, wherein n=1,2,3,4 or 5 (R is-C
3F
7The time ligand be abbreviated as BHHCT);
Described trivalent europium ion, four tooth beta-diketon class ligand and 2-(4-N, N-diethylbenzene amido)-4,6-two (3,5-dimethyl pyrazole base)-1,3,5-triazine, the molar ratio between them are 1: 0.5～3: 0.5～3, and to keep the molar ratio of four tooth beta-diketon class ligands and aminosilane reagent be 1: 1～4;
The molecular formula of described silicon ester is Si (OR)
4, R wherein is-C
nH
2n+1, n=1～5.
Described aminosilane reagent and have amino silanization monomer and all can be 3-aminopropyl tri-alkoxy silicon or N-(β-aminoethyl)-gamma-aminopropyl-triethoxy-silicon, the silanization monomer that has sulfydryl is a 3-sulfydryl propyl trialkoxy silicon.
The concrete preparation process of excited by visible light nanometer europium fluorescent particle is:
1) fluorescence precursor: in organic solvent, earlier four tooth beta-diketon class ligands and aminosilane reagent were stirred 0.5～4 hour at ambient temperature, make it to take place the covalent attachment reaction, both molar ratios are 1: 1～4, the volumetric molar concentration of four tooth beta-diketon class ligands is 1～15 μ mol/ml in the system, and then adds Eu
3+And 2-(4-N, N-diethylbenzene amido)-4,6-two (3 base)-1,3,5-triazines continues to stir 4-24 hour, keeps Eu
3+, four tooth beta-diketon class ligands and 2-(4-N, N-diethylbenzene amido)-4, the molar ratio of 6-two (3 base)-1,3,5-triazines is 1: 0.5～3: 0.5～3, evaporating solvent and the dry fluorescence precursor that obtains excited by visible light;
2) preparation microemulsion: with organic solvent, cosurfactant, tensio-active agent and hydromassage you than being 15～25: 2～7: make microemulsion behind 1: 6～10 uniform mixing;
3) in microemulsion, add the fluorescence precursor, room temperature condition stirred 0.5～1 hour down, added silicon ester and the silanization monomer that has active group amino or sulfydryl then, with the ammoniacal liquor initiated polymerization of weight content 3-25%, room temperature reaction is 4～48 hours under stirring, and centrifugation gets final product.
The concentration of fluorescence precursor in microemulsion system is 0.05～1mg/ml;
Silicon ester is 1: 0.05～0.2 with the monomeric mol ratio of silanization that has active group amino or sulfydryl, and the volumetric molar concentration of silicon ester in microemulsion system is 14～45 μ mol/ml.
Described organic solvent is: benzene, normal hexane, hexanaphthene or sherwood oil; Cosurfactant is ethanol, propyl alcohol, Virahol, n-hexyl alcohol, ethylene glycol, n-Octanol or nonylcarbinol; Tensio-active agent is polyoxyethylene groups octyl phenyl ether or polyoxyethylene groups nonylplenyl ether.
In a word, the present invention is a luminophore with excited by visible light hyperfluorescence ternary europium complex, adopt the method for covalent bonding the rare-earth fluorescent title complex to be securely fixed in the network system of silica gel, be prepared from by the synthetic grain of a Reversed Phase Micro Emulsion Copolymerization technology, and nano-particle surface introduce simultaneously can with the active function groups of biomolecules covalent bonds.
Excited by visible light nanometer europium fluorescent particle of the present invention can be used as marker and is used for various time resolved fluorescence bioanalysiss, comprise the time resolved fluoro-immunoassay method, time resolved fluorescence DNA hybridization analysis method, time resolved fluorescence microscope imaging analytical method and time are differentiated the fluorescent biochip analytical method.
The nanometer europium fluorescent particle of the present invention thing that serves as a mark has following advantage:
(1) realized having the long fluorescence lifetime biomarker of visible excitation peak.In the aqueous solution, nanometer europium fluorescent particle of the present invention still has strong excitation peak at visible region, and its excitation spectrum may extend into the above blue light region of 450nm.
(2) significantly improved the fluorescence radiation intensity of nano fluorescent bioprobe.The novel tertiary europium complex presoma itself that the present invention adopts has very strong fluorescence radiation intensity, adopt the method for this presoma and silicon ester copolymerization to make and be fixed with a large amount of europium complexes in the single nanoparticle, greatly improved the fluorescence radiation intensity of nano fluorescent particulate.
(3) light stability strengthens.For the rare-earth fluorescent compound of most ultraviolet excitations; low-energy excited by visible light significantly improves the anti-photobleaching ability of nanometer europium fluorescent grain; can prolong the METHOD FOR CONTINUOUS DETERMINATION time of marker; simultaneously; outer silica matrix can effectively shield solvent and living radical on every side, further the inner europium complex of protection.
(4) the present invention has set up the time resolved fluorescence micro-imaging analyzing novel methods based on the highly sensitive pathogeny microorganism of excited by visible light nanometer europium fluorescent marker.This method not only can be eliminated the interference of various short life background fluorescences to measuring in the complex environment sample fully, make that specificity and the sensitivity measured are greatly improved, and, indicated that novel method also should have good application prospects at viable cell and biological tissue time resolved fluorescence micro-imaging mensuration field because the time-resolved fluorometry of excited by visible light can be avoided the damage of exciting light to biological sample.
Description of drawings
Fig. 1 is the transmission electron microscope photo of excited by visible light nanometer europium fluorescent grain.A, b, c, d, e are respectively the nanometer europium fluorescent grain that obtains when adding difference amount (2,4,6,8,10 milligrams) fluorescence presoma.
Fig. 2 is fluorescence precursor A PS-BHHCT-Eu
3+The time resolved fluorescence excitation spectrum of-DPBT in different solvents.A, N, dinethylformamide; B, tetrahydrofuran (THF); C, ethanol; D, acetone; E, normal heptane; F, toluene; G, chloroform; H, methylene dichloride.
Fig. 3 is the time resolution fluorescence spectral of excited by visible light nanometer europium fluorescent grain in the Tris-HCl of pH value 7.8 buffered soln.
Fig. 4 is the uv-visible absorption spectra of excited by visible light nanometer europium fluorescent grain (A) and pure silicon glue nanoparticle (B).
Fig. 5 is the light stability experimental result of excited by visible light nanometer europium fluorescent grain (A) and rhodamine B (B).
Fig. 6 is the working curve with the prostate specific antigen (PSA) in the plain time-resolved fluorescent immunoassay human serum of excited by visible light nanometer europium fluorescent grain mark strepto-affinity.
Fig. 7 is an imaging picture of measuring pathogeny microorganism Giardia lamblia in the ambient water sample with the plain time resolved fluorescence micro-imaging of excited by visible light nanometer europium fluorescent grain mark strepto-affinity.(a) light field imaging; (b) 330-380nm excites conventional fluorescence imaging; (c) 380-420nm excites conventional fluorescence imaging; (d) the 330-380nm firing time is differentiated fluorescence imaging; (e) the 380-420nm firing time is differentiated fluorescence imaging.
Fig. 8. measure the microscope imaging picture of Giardia lamblia control experiment in the ambient water with the plain time resolved fluorescence micro-imaging of excited by visible light nanometer europium fluorescent grain mark strepto-affinity.(a) light field imaging; (b) 330-380nm excites conventional fluorescence imaging; (c) 380-420nm excites conventional fluorescence imaging; (d) the 330-380nm firing time is differentiated fluorescence imaging.
Fig. 9 is in the light stability measuring result of microscopically behind the excited by visible light nanometer europium fluorescent grain specific recognition Giardia lamblia.(A) 5 minutes time resolved fluorescence picture (the 330-380nm xenon lamp excites) of irradiation under mercury lamp 330-380nm wave band high light; (B) 5 minutes time resolved fluorescence picture (the 380-420nm xenon lamp excites) of irradiation under mercury lamp 380-420nm wave band high light.
Embodiment
The invention will be further described below by embodiment.Present embodiment only is used for that the present invention will be described, also belongs to scope of the present invention based on the method for same principle and similar raw material.
Embodiment 1: the preparation and the sign of excited by visible light nanometer europium fluorescent grain
(1) fluorescence precursor A PS-BHHCT-Eu
3+The preparation of-DPBT
25 μ mol BHHCT are dissolved in the 4mL anhydrous tetrahydro furan, stir and add 62.5 μ mol aminopropyl triethoxysilanes (APS) down, stirring at room 3 hours adds the anhydrous tetrahydrofuran solution that 15mL is dissolved with 50 μ molDPBT then, stirs the back a little and adds 25 μ mol EuCl
36H
2O stirred 24 hours, with Rotary Evaporators solvent evaporate to dryness and vacuum-drying was promptly got fluorescence precursor A PS-BHHCT-Eu
3+-DPBT.
(2) preparation of excited by visible light nanometer europium biological probe
With above-mentioned fluorescence precursor A PS-BHHCT-Eu
3+(2 milligrams of-DPBT, 4 milligrams, 6 milligrams, 8 milligrams and 10 milligrams) be dissolved in the 1.5mL toluene, join respectively in the reverse micro emulsion that contains 7.25 gram hexanaphthenes, 2.37 gram Triton X-100,1.82 gram n-Octanols and 0.55mL water, stirring at room 15 minutes adds 100 μ L tetraethoxys (TEOS) and 5 μ LAPS then, adds 100 μ L ammoniacal liquor initiated polymerizations again.Behind the reaction solution room temperature reaction 24 hours, add ethanol the solid nano particulate deposits is come out, centrifugal back is with ethanol and washing, to remove unreacted raw material and tensio-active agent.
(3) form of nanometer europium fluorescent particle and size dimension characterize
Fig. 1 is the transmission electron microscope photo of excited by visible light nanometer europium fluorescent grain, is the 2-8 milligram hour from scheming as seen to work as the presoma add-on, and nano-fluorescent grain is uniform spheroidal, and particle diameter is all less than 50nm, and monodispersity is good.Continue to increase presoma add-on to 10 milligram, then the nano-fluorescent grain surface becomes coarse and occurs assembling.In addition, the amount of the concentration of the kind of the ratio of amount, water and the tensio-active agent that the size of particle diameter can be by adjusting presoma, cosurfactant, ammoniacal liquor, silicon ester (as TEOS) and reaction times are waited and control.
(4) fluorescence precursor A PS-BHHCT-Eu
3+The fluorescence spectrum characteristic of-DPBT
Fig. 2 is fluorescence precursor A PS-BHHCT-Eu
3+The time resolved fluorescence excitation spectrum of-DPBT in different organic solvents.As seen from the figure in the little non-corrdination type solvent of polarity (as toluene, chloroform, normal heptane and methylene dichloride etc.), presoma is very strong at the visible excitation peak at 406nm place, and in strong polarity corrdination type solvent (as N, dinethylformamide, tetrahydrofuran (THF), ethanol and acetone etc.), as seen excitation peak seriously weakens even disappears, and visible solvent property is to fluorescence precursor A PS-BHHCT-Eu
3+The fluorescence excitation character of-DPBT has very big influence.
(5) the fluorescence spectrum characteristic of nanometer europium fluorescent grain
Fig. 3 is the time resolution fluorescence spectral of nanometer europium fluorescent grain in the Tris-HCl of pH value 7.8 buffered soln.Nano particle still has the visible excitation peak of very strong 406nm in buffered soln as seen from the figure, illustrates that nanometer europium fluorescent grain that this preparation method obtains has kept the excited by visible light characteristic of fluorescence presoma to a great extent.In addition, the fluorescence lifetime of the nano-fluorescent grain that this method makes is 0.39 millisecond after measured, and the fluorescence that the nano fluorescent particulate is described has still kept the long fluorescence lifetime feature of europium complex.
(6) uv-visible absorption spectra of nanometer europium fluorescent grain and pure silicon glue nano particle
As Fig. 4, compare with the uv-visible absorption spectra figure of pure silicon glue nano particle, several tangible absorption peaks have appearred in nanometer europium fluorescent grain, are included in 416nm the wide absorption peak of the visible region that is the center, illustrate that the fluorescence presoma has been attached in the middle of the silica matrix effectively.
(7) light stability of nanometer europium fluorescent grain experiment
The light stability experimental result of nanometer europium fluorescent grain as shown in Figure 5, used light source is the 30W deuterium lamp.At strong illumination that deuterium lamp sends after 60 minutes, the fluorescence intensity of organic fluorescent compounds rhodamine B decays to for 32% when initial, and the fluorescence intensity of nanometer europium fluorescent grain only decays to for 70% when initial, illustrates that the nanometer europium fluorescent grain of making has the light stability that is much better than organic fluorescent compounds.
Embodiment 2: the nanometer europium fluorescent grain thing that serves as a mark is used for time-resolved fluorescent immunoassay human prostate specific antigen (be called for short PSA)
(1) nanometer europium fluorescent particle labelled streptavidin (being called for short SA)
With 1.0 milligrams of nanometer europium fluorescent grains and 3.0 milligrams of bovine serum albumins (BSA) 0.1molL with pH value 7.1
-1After phosphate buffer solution 1.2mL dissolves fully, stir 1% glutaraldehyde water solution that adds 0.3mL down, stirring at room 24 hours.With the centrifugal collection of nano particle and with behind the phosphate buffer solution thorough washing, add 0.9mL and contain the phosphate buffer solution of 0.2 milligram of SA and 1% glutaraldehyde water solution of 0.2mL, 4 ℃ were stirred 22 hours, added 2.0 milligrams of sodium borohydrides and continued reaction 2 hours.The nano particle that the surface indicates the SA molecule is behind centrifugal, phosphate buffer solution thorough washing, with the 0.05molL of pH value 8.0
-1Ammonium bicarbonate aqueous solution is an elutriant, is further purified with the SephadexG-50 post of 1.0 * 29.1cm, obtains the SA solution of 2.6mL nanoparticle label.Add 5.2 milligrams BSA and 2.6 milligrams sodiumazide, 4 ℃ of preservations are standby.When being used for immunoassay, with containing 0.2%BSA-0.1%NaN
3The 0.05molL of the pH value 7.8 of-0.9%NaCl
-1The dilution of Tris-HCl buffered soln.
(2) preparation of the anti-people PSA of biotin labeling goat antibody
1.0mL the anti-people PSA of goat antibody (0.5mgmL
-1) 4 ℃, 24 hours to twice dialysis of 3L water after, add 8.4 milligrams sodium bicarbonate and 3 milligrams NHS-LC-biotin, stirring at room is after 1 hour, places in following 24 hours for 4 ℃.Reaction solution adds 5 milligrams BSA and 5 milligrams sodiumazide after 3L being contained twice dialysis of 0.1mol/L sodium bicarbonate aqueous solution of 0.25 gram sodiumazide in 4 ℃, 24 hours, place-20 ℃ standby.When being used for immunoassay, after diluting 300 times, uses by usefulness buffered soln 1.
(3) time-resolved fluorescent immunoassay people PSA
(a) the bag quilt of 96 microwell plates: in each hole of 96 microwell plates, add 50 μ L respectively and contain 10 μ gmL
-1The 0.1molL of the pH value 9.6 of mouse anti human PSA monoclonal antibody
-1Sodium bicarbonate buffer solution, 4 ℃, 24 hours bags by after, be 7.8 0.05molL with the pH value that contains 0.05%Tween 20
-1Tris-HCl buffered soln (buffered soln 1) washes twice, is 7.8 0.05molL again with the pH value
-1Tris-HCl buffered soln (buffered soln 2) is washed 1 time, ℃ preservation down of the microwell plate-20 behind the bag quilt.
(b) immunoassay of people PSA: with PSA antigen standardized solution with containing 5%BSA-0.9%NaCl-0.1%NaN
3The pH value be 7.8 0.05molL
-1Tris-HCl buffered soln is diluted to finite concentration, get standardized solution 45 μ L inject above-mentioned bag by after each hole of microwell plate, after the reaction in 37 ℃, 1 hour, wash twice with above-mentioned buffered soln 1, buffered soln 2 is washed once.The biotin labeled anti-PSA antibody-solutions of 45 μ L is joined in each micropore, after the reaction in 37 ℃, 1 hour, wash twice with above-mentioned buffered soln 1, buffered soln 2 is washed once.The SA solution of 45 μ L nanometer particle to mark is joined in each micropore, after the reaction in 37 ℃, 1 hour, wash 4 times, carry out the solid phase time-resolved fluorometry then with buffered soln 1.Measuring with instrument is Perkin Elmer Victor 1420 multiple labeling calculating instruments, and condition determination is: excitation wavelength, 340nm; Detect wavelength, 615nm; Dead time, 0.2ms; The window time, 0.4ms; Cycling time, 1.0ms.
The working curve of measuring people PSA with this method as shown in Figure 6, this law minimum detects down and is limited to 29.8pg/mL (method of calculation: dl=(3SD * C)/(I-I
0), SD is the standard deviation of background signal, and C is minimum mensuration liquid concentration, and I is its corresponding fluorescence intensity, I
0Be the fluorescence intensity of background signal), the working curve upper limit can reach 50ng/mL.
Embodiment 3: the nanometer europium fluorescent grain thing that serves as a mark is used for pathogeny microorganism-Giardia lamblia (Giardia lamblia) that the time resolved fluorescence microscope imaging is measured the complex environment sample
(1) the time resolved fluorescence microscope imaging of Giardia lamblia is measured in the complex environment sample
Get 5 μ L Giardia lamblias (2 * 10
6Individual/mL) joining 15 μ L contains in 10000 times of concentrated ambient water samples of a large amount of various impurity, add the anti-Giardia lamblia monoclonal antibody of 16 μ L (50 μ g/mL) then successively, the anti-mouse secondary antibodies of the biotin labeled rabbit of 16 μ L (50 μ g/mL, the preparation method is with the anti-people PSA of biotin labeling goat antibody) and the SA solution of 6 μ L nano-fluorescent grain marks, incubated at room 24 hours, low-speed centrifugal (500 rev/mins) is collected Giardia lamblia and is dispersed in again in the water, removes unreacted nanoparticle and antibody repeatedly several times.Giardia lamblia behind the nanoparticle immunostaining is diluted with ambient water, take a morsel then to place and carry out microscope imaging mensuration on the wave carrier piece.
Fig. 7 is the microscope imaging measurement result of Giardia lamblia in the ambient water.As seen from the figure, the background fluorescence that exists coexistent impurity in the very strong sample in the conventional fluorescence imaging picture that records with the exciting light of 330-380nm and 380-420nm disturbs respectively, and the background fluorescence of impurity is eliminated fully in the picture that employing time resolved fluorescence imaging pattern records, only can be observed by the danger signal of the Giardia lamblia of nano particle specific stain, description time is differentiated the fluorescence imaging pattern and is had high selectivity and sensitivity.In addition, contrast two fluorescence imaging pictures that excite wave band (330-380nm and 380-420nm) as can be seen, fluorescence signal intensity under purple light (380-420nm) excites only is weaker than the fluorescence signal intensity of UV-light (330-380nm) under exciting slightly, is enough to satisfy the needs that the time resolved fluorescence micro-imaging is measured.
(2) specificity-control experiment of immunostaining and imaging mensuration
5 μ L Giardia lamblias (2 * 10
6Individual/as to add the SA solution of anti-mouse secondary antibodies of the biotin labeled rabbit of 16 μ L (50 μ g/mL) and 6 μ L namo fluorescence probe marks in mL), incubated at room 24 hours, low-speed centrifugal (500 rev/mins) is collected Giardia lamblia and is dispersed in the water again, remove unreacted nanoparticle and antibody repeatedly several times, with the dilution of secondary water, take a morsel to place and carry out microscope imaging mensuration on the wave carrier piece at last.
Fig. 8 is the microscope imaging measurement result of Giardia lamblia under this reaction conditions.As seen from the figure under the situation that does not add anti-Giardia lamblia monoclonal antibody, the Giardia lamblia surface does not have the red fluorescence of nanoparticle, illustrates that nano-fluorescent grain can not be adsorbed on the surface of Giardia lamblia non-specificly and produce false-positive result under this condition.
(3) light stability of the nanometer europium fluorescent marker on Giardia lamblia surface
The painted Giardia lamblia of nanometer europium fluorescent grain specific immunity was shone respectively 5 minutes with the 330-380nm and the 380-420nm band of light of the 100W mercury lamp of microscope equipment, gathered a time resolved fluorescence imaging picture every one minute, the light stability of observing the nanometer europium fluorescent marker on Giardia lamblia surface.
Fig. 9 has provided the experimental result under this condition.As seen from the figure, under high-octane 330-380nm rayed, the red fluorescence bleaching speed of the nanometer europium fluorescent marker on Giardia lamblia surface is very fast, substantially, completely dissolve during by 5 minutes, and under low-energy 330-380nm rayed, the red fluorescence bleaching speed of nanometer europium fluorescent marker is obviously slack-off, and the red fluorescence signal is still high-visible after 5 minutes.This result has convincingly demonstrated the photobleaching speed that excited by visible light can significantly weaken marker, thereby but prolonging METHOD FOR CONTINUOUS DETERMINATION time of sample, this advantage should have good using value in some needs the research of long-time continuous tracer determination.
Claims (8)
1. the nanometer europium fluorescent particle that has the excited by visible light performance, it is characterized in that: it is that the covalent bonding reaction takes place in organic solvent for ternary europium complex by aminosilane reagent and a kind of excited by visible light, obtain the fluorescence presoma, it forms the functional nano europium fluorescent particle that has the excited by visible light performance and can be used for biomarker again with silicon ester and have the silanization monomer generation copolymerization of active group amino or sulfydryl then;
The ternary europium complex of wherein said excited by visible light is by trivalent europium ion and four tooth beta-diketon class ligand and 2-(4-N, N-diethylbenzene amido)-4,6-two (3,5-dimethyl pyrazole base)-1,3, the ternary fluorescence complex that 5-triazine ligand forms, the structural formula of two kinds of ligands is:
R is=C in the formula
6F
5Or fat hydrocarbon substituting group C
nH
2n+1Or C
nF
2n+1, n=1,2,3,4 or 5 wherein;
Described trivalent europium ion, four tooth beta-diketon class ligand and 2-(4-N, N-diethylbenzene amido)-4,6-two (3,5-dimethyl pyrazole base)-1,3,5-triazine, the molar ratio between them are 1: 0.5～3: 0.5～3, and to keep the molar ratio of four tooth beta-diketon class ligands and aminosilane reagent be 1: 1～4.
2. nanometer europium fluorescent particle according to claim 1 is characterized in that: the molecular formula of described silicon ester is Si (OR)
4, R wherein is-C
nH
2n+1, n=1～5.
3. nanometer europium fluorescent particle according to claim 1, it is characterized in that: described aminosilane reagent and have amino silanization monomer and all can be 3-aminopropyl tri-alkoxy silicon or N-(β-aminoethyl)-gamma-aminopropyl-triethoxy-silicon, the silanization monomer that has sulfydryl is a 3-sulfydryl propyl trialkoxy silicon.
4. the preparation method of the described nanometer europium of claim 1 fluorescent particle is characterized in that:
Specific operation process is,
1) fluorescence precursor: in organic solvent, earlier four tooth beta-diketon class ligands and aminosilane reagent were stirred 0.5～4 hour at ambient temperature, make it to take place the covalent attachment reaction, both molar ratios are 1: 1～5, the volumetric molar concentration of four tooth beta-diketon class ligands is 1～15 μ mol/ml in the system, and then adds Eu
3+And 2-(4-N, N-diethylbenzene amido)-4,6-two (3 base)-1,3,5-triazines continues to stir 4～24 hours, keeps Eu
3+, four tooth beta-diketon class ligands and 2-(4-N, N-diethylbenzene amido)-4, the molar ratio of 6-two (3 base)-1,3,5-triazines is 1: 0.5～3: 0.5～3, evaporating solvent and the dry fluorescence precursor that obtains excited by visible light;
2) preparation microemulsion: with organic solvent, cosurfactant, tensio-active agent and hydromassage you than being 15～25: 2～7: make microemulsion behind 1: 6～10 uniform mixing;
3) in microemulsion, add the fluorescence precursor, room temperature condition stirred 0.5～1 hour down, add silicon ester and the silanization monomer that has active group amino or sulfydryl then, ammoniacal liquor initiated polymerization with weight content 3-25%, room temperature reaction is 4～48 hours under stirring, centrifugation gets final product, thus the fluorescent nano particles of preparing excited by visible light and having the biomarker function;
The concentration of fluorescence precursor in microemulsion system is 0.05～1mg/ml;
Silicon ester is 1: 0.05～0.2 with the monomeric mol ratio of silanization that has active group amino or sulfydryl, and the volumetric molar concentration of silicon ester in microemulsion system is 14～45 μ mol/ml.
5. according to the preparation method of the described nanometer europium of claim 4 fluorescent particle, it is characterized in that:
Described organic solvent is: benzene, normal hexane, hexanaphthene or sherwood oil; Cosurfactant is ethanol, propyl alcohol, Virahol, n-hexyl alcohol, ethylene glycol, n-Octanol or nonylcarbinol; Tensio-active agent is polyoxyethylene groups octyl phenyl ether or polyoxyethylene groups nonylplenyl ether.
6. according to the preparation method of the described nanometer europium of claim 4 fluorescent particle, it is characterized in that:
Step 1) and 3) aminosilane reagent described in and have amino silanization monomer and all can be 3-aminopropyl tri-alkoxy silicon or N-(β-aminoethyl)-gamma-aminopropyl-triethoxy-silicon;
The silanization monomer that has sulfydryl described in the step 3) is a 3-sulfydryl propyl trialkoxy silicon.
7. the described nanometer europium of claim 1 fluorescent grain is as the application of biomarker, and it is characterized in that: described nanometer europium fluorescent grain utilizes the time-resolved fluorometry mode to carry out biochemical measurement as nano-probe.
8. according to the described application of claim 7, it is characterized in that: described time-resolved fluorometry mode is meant time-resolved fluorescent immunoassay, time resolved fluorescence DNA hybridization assays method, time resolved fluorescence microscope imaging assay method, time resolved fluorescence cytoactive assay method or time resolved fluorescence biochip assay method.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103175953A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Europium nanoparticle fluorescence quality control serum as well as preparation method and application thereof |
| CN103278481A (en) * | 2012-01-02 | 2013-09-04 | 何爱民 | Binding assays utilizing time-resolved up-converting luminescence detection |
| CN103293133A (en) * | 2012-01-02 | 2013-09-11 | 何爱民 | Magnetic binding assays method utilizing time-resolved up-converting luminescence detection |
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| CN1482459A (en) * | 2002-09-11 | 2004-03-17 | 中国科学院大连化学物理研究所 | Trivalent europium-beta-diketone fluorescent label and uses thereof |
| CN1566954A (en) * | 2003-07-04 | 2005-01-19 | 中国科学院大连化学物理研究所 | Beta-diketone-trivalent europium complex nano fluorescent probe, its preparation and use thereof |
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| CN1482459A (en) * | 2002-09-11 | 2004-03-17 | 中国科学院大连化学物理研究所 | Trivalent europium-beta-diketone fluorescent label and uses thereof |
| CN1566954A (en) * | 2003-07-04 | 2005-01-19 | 中国科学院大连化学物理研究所 | Beta-diketone-trivalent europium complex nano fluorescent probe, its preparation and use thereof |
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| CN103175953A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Europium nanoparticle fluorescence quality control serum as well as preparation method and application thereof |
| CN103175953B (en) * | 2011-12-26 | 2015-04-15 | 苏州新波生物技术有限公司 | Europium nanoparticle fluorescence quality control serum as well as preparation method and application thereof |
| CN103278481A (en) * | 2012-01-02 | 2013-09-04 | 何爱民 | Binding assays utilizing time-resolved up-converting luminescence detection |
| CN103293133A (en) * | 2012-01-02 | 2013-09-11 | 何爱民 | Magnetic binding assays method utilizing time-resolved up-converting luminescence detection |
| CN103293133B (en) * | 2012-01-02 | 2016-04-20 | 何爱民 | Time resolution up-conversion luminescence detection technique is utilized to carry out magnetic in conjunction with method for measuring |
| CN107037025A (en) * | 2017-06-24 | 2017-08-11 | 杭州微瑞科技有限公司 | The high quick fluorescent chromatographic device of group of the lanthanides and detection method |
| CN112888947A (en) * | 2018-07-18 | 2021-06-01 | 巴黎综合理工大学 | Capillary action test using photoluminescent inorganic nanoparticles |
| CN112414989A (en) * | 2020-12-16 | 2021-02-26 | 江南大学 | Method for detecting melamine by taking tetracycline hydrochloride-europium as fluorescent probe |
| CN112414989B (en) * | 2020-12-16 | 2022-01-07 | 江南大学 | Method for detecting melamine by taking tetracycline hydrochloride-europium as fluorescent probe |
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